B: DNA replication Flashcards
What is the difference between Go, G1, G2, S and M phases of the cell cycle?
G1 = cell grows and prepares for cell division, checks cell cycle checkpoints - if they are not approved cell stops growing.
G0 = non-dividing state
S = DNA replication phase
G2 = Cell prepares for mitosis by growing, increasing organelles, producing proteins.
M = Mitosis + cell division
What is an origin of replication?
Point on DNA where replication begins.
What is the difference between NTPs and dNTPs?
NTPs have ribose as sugar, dNTPs have deoxyribose
List the items required by DNA polymerase for DNA synthesis
- dNTPS
- Primer
- DNA phosphate
- Template
Difference between DNA and RNA polymerase?
DNA polymerase produces double stranded DNA molecule, RNA polymerase produces single stranded molecule.
What is a primer?
short existing piece of DNA/RNA that allows DNA polymerase to make new DNA
What is the direction of DNA synthesis?
5’ to 3’
What is the basic DNA polymerase error rate?
1x10-5 = in vitro (not compatible with human life)
1x10-10 = in vivo (actual number)
Describe proof reading by DNA polymerase
Carried out by 3’-5’ exonuclease in DNA polymerase, digests DNA starting at 3’ and moving towards 5’.
A misincorporated nucleotide will slow down polymerisation and increase activity of exonuclease.
3’-5’ exonuclease excises misincorporated nucleotide and DNA polymerase then resynthesises excised section and continues on.
Describe mismatch repair
Sometimes a misincorporated nucleotide is not excised during proofreading and creates a mismatched DNA pair.
This is recognised by DNA mismatch repair system and corrected, mismatch repair system must be able to distinguish between template + new strand of DNA to remove mismatched base.
Explain the differences between DNA polymerase proof reading and mismatch repair
Proofreading corrects errors during DNA replication, mismatch repair corrects errors after replication.
What is HNPCC and what are the characteristics of HNPCC + genes that cause it
Hereditary Nonpolyposis colorectal cancer
Amsterdam criteria:
- 3 relatives over 2 generations with colorectal cancer
- 2 must be first degree relatives
- 1 must be under 50 at time of diagnosis
- FAP (familial adenomotous polyposis) must be excluded
Mutations in MMR genes that cause HNPCC:
- MLH1
- MSH2
- MSH3
- MSH6
- PMS1
- PMS2
Explain how DNA replication is initiated
- Recognition: Origin Recognition Complex (ORC proteins) bind to origin of replication
- Melting: Proteins are ori recruit additional proteins and melt the DNA
- Unwinding: double helix needs to be unwound -carried out by helicases to form a replication bubble at ori. Single stranded binding protein binds to DNA to keep it single stranded so various enzymes can work on it.
- Recruitment: ori will recruit proteins that replicate the DNA
- Primer synthesis: RNA primer is synthesised at ori by DNA primase. Supercoiling needs to be relieved by topoisomerase. Once primer is synthesised, DNA polymerase can start DNA synthesis in 5’-3’ direction.
Eytan:
- Recognition: ORC in eukaryotes, DnaA in prokaryotes recgognize and bind the ori
- Melting: of DNA at ori by additional recruited proteins
- Unwinding: helicases form a replication bubble at the ori to make room for initiation proteins to access the DNA at the ori
- Recruitment: proteins at ori recruit proteins that will replicate DNA (including DNA polymerase)
- Primer synthesis: DNA primase synthesizes RNA primer at ori; supercoiling relieved by topoisomerases 1 and 2 in human, DNA gyrase in prokaryotes
- DNA synthesis: by DNA polymerase in a 5’-3’ direction
Chemotherapeutic agent etoposide inhibits:
DNA topoisomerase
Silent mutation
Change of codon, but still codes for same amino acid –> usually no effect
Missense mutation
Change of codon, codes for different amino acid –> may have major, minor or no effect
Nonsense mutation
Change of codon, codes for STOP codon –> usually major effect (kills function of protein)
Fen-1 and RNAseHI
5’-3’ exonuclease removes RNA primer
Etoposide, anthracyclines, mitoxantrone?
inhibit topoisomerase II and cause cell death –> anti-cancer agents
Fluoroquinolones + aminocoumarin?
inhibit DNA gyrase + kill bacteria –> antibiotics
What does a telomere do?
Extends 3’ end by TTAGGG
Enzymes in E.coli and human responsible for DNA unwinding
DNA helicase in both
Enzymes in E.coli and human responsible for primer synthesis
DNA primase in both
Enzymes in E.coli and human responsible for main DNA synthesis
E. coli = DNA polymerase 3
Human = DNA polymerase alpha/epsilon
Enzymes in E.coli and human responsible for RNA primer removal
E. coli = DNA polymerase 1 (5’-3’ exonuclease)
Human = Fen1 and RNAseH1 (5’-3’ exonuclease)
Enzymes in E.coli and human responsible for relieving coiling
E. coli = DNA gyrase
Human = Topoisomerase II
Enzymes in E.coli and human responsible for binding DNA together
DNA ligase in both
Typical start codon?
AUG
Stop codons?
UGA, UAG, UAA
How does DNA replication terminate in E.Coli
Genome is circular - replication fork proceeds around circle to a termination site opposite ori.
Contains 10 ter sequences, tus protein interacts with ter sequences to initiate termination.
Completed replication genomes are intertwined and relieved by topoisomerase IV.
How DNA replication terminate in eukaryotes
Telomere
